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Offlinemetaphoric
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Tissue cloning sterile technique question
    #26566579 - 03/30/20 05:43 AM (3 years, 9 months ago)

Hey folks. I'm about to make my second attempt cloning a fruit. Last time I used a sharp knife and didn't love it. This time I want to use mini scissors, like the kind for trimming nose hairs. They are just the right size. Only thing is I don't want to flame sterilize. Can I just leave it soaking in rubbing alcohol in my SAB? I would pick the scissors up out of the alcohol and perform the operation while they're still wet.


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Onlinerumfor69
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Re: Tissue cloning sterile technique question [Re: metaphoric]
    #26566597 - 03/30/20 06:16 AM (3 years, 9 months ago)

If they're metal you could just do 15mins @15psi in a jar with a lid.

I dunno if the alcohol alone will be enough, flame sterilizing the
Tips would be exactly what you would wanna do with a scalpel so I
wouldn't reccomend anything else really.

Are you planning on taking tissue from the inside of the stem?


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Invisiblerickyswamps
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Re: Tissue cloning sterile technique question [Re: rumfor69]
    #26566600 - 03/30/20 06:20 AM (3 years, 9 months ago)

Why not flame them?


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Offlinemetaphoric
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Re: Tissue cloning sterile technique question [Re: rumfor69]
    #26566602 - 03/30/20 06:21 AM (3 years, 9 months ago)

Yeah, gloves on, tear the stem open with my fingers and snip/ transfer a little piece of tissue from the inside.

The mushroom was also grown in vitro so hopefully all goes smooth 👍👍


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:mushroom:                  :mushroom2:                :mushroom:
:mushroom:            :mushroom2::mushroom2::mushroom2:            :mushroom:
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OfflineQave
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Re: Tissue cloning sterile technique question [Re: metaphoric]
    #26566627 - 03/30/20 06:43 AM (3 years, 9 months ago)

Pressure cooker sterilizing will 100% work for the scissors, but technically they aren't sterile after the first use.

If you're open to alternatives, I've used creamcorn's needle sample tek and really liked it.


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Offlinemetaphoric
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Re: Tissue cloning sterile technique question [Re: rickyswamps]
    #26566665 - 03/30/20 07:12 AM (3 years, 9 months ago)

Quote:

rickyswamps said:
Why not flame them?




I don't want to make the scissors all black until I test it out first. If I like it as a tool I'll buy another one just for mycology.


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:mushroom:            :mushroom2::mushroom2::mushroom2:            :mushroom:
:mushroom:      :mushroom2::mushroom2::mushroom2::mushroom2::mushroom2:      :mushroom:
:mushroom::cookiemonster: BEHOLD :elmo::mushroom:
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Invisibletramalot
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Re: Tissue cloning sterile technique question [Re: metaphoric]
    #26566682 - 03/30/20 07:31 AM (3 years, 9 months ago)

Just grab a piece with tweezers?


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Offlinemetaphoric
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Re: Tissue cloning sterile technique question [Re: Qave]
    #26566683 - 03/30/20 07:32 AM (3 years, 9 months ago)

Quote:

Qave said:
Pressure cooker sterilizing will 100% work for the scissors, but technically they aren't sterile after the first use.

If you're open to alternatives, I've used creamcorn's needle sample tek and really liked it.




Wow! I just finished reading that, I like it a lot, thanks for sharing.

How many times have you used this tek and have you ever had it contaminate? I've stayed away from LCs so far because I hate how you don't know if you're contaminated until months later when you try to fruit.


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:mushroom::mushroom::mushroom::mushroom::mushroom::mushroom::mushroom::mushroom::mushroom::mushroom:
:mushroom:                  :mushroom2:                :mushroom:
:mushroom:            :mushroom2::mushroom2::mushroom2:            :mushroom:
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Offlinemetaphoric
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Re: Tissue cloning sterile technique question [Re: tramalot]
    #26566684 - 03/30/20 07:33 AM (3 years, 9 months ago)

Quote:

tramalot said:
Just grab a piece with tweezers?



I don't have tweezers but yeah, that's why I like these little scissors, I can use them like tweezers after snipping a piece free.


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:mushroom::mushroom::mushroom::mushroom::mushroom::mushroom::mushroom::mushroom::mushroom::mushroom:
:mushroom:                  :mushroom2:                :mushroom:
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:mushroom:      :mushroom2::mushroom2::mushroom2::mushroom2::mushroom2:      :mushroom:
:mushroom::cookiemonster: BEHOLD :elmo::mushroom:
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OfflineA.k.aM
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Re: Tissue cloning sterile technique question [Re: metaphoric]
    #26566761 - 03/30/20 08:38 AM (3 years, 9 months ago)

I’ve found it easiest to cut down diagonally into the stem to the right then another to the left so you’ve cut a V into the flesh, then either take the whole piece or slice the top layer of the V off depending how deep of an angle you did.

Definitely need a fresh sharp blade but it’s the easiest method I’ve found so far.

Edit: this is using half the shroom, after pulling the stem apart. Cutting into the outside will not go well.


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Edited by A.k.a (03/30/20 09:14 AM)


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OfflineBallzagna
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Re: Tissue cloning sterile technique question [Re: A.k.a]
    #26566794 - 03/30/20 08:54 AM (3 years, 9 months ago)

Do you flame between cuts? Even so, I've read that cutting it can force contams  into the clean flesh inside which is why Frank's cloning tech recommends tearing it


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OfflineA.k.aM
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Re: Tissue cloning sterile technique question [Re: Ballzagna]
    #26566840 - 03/30/20 09:13 AM (3 years, 9 months ago)

I meant after you’ve torn it open. Cutting into it from outside will definitely contaminate it.

I don’t flame at all for cloning, I tear it in half then use a new blade cuz it’s a pain in the ass with a dull one.


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OfflineMooshi97
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Re: Tissue cloning sterile technique question [Re: A.k.a]
    #26566964 - 03/30/20 10:38 AM (3 years, 9 months ago)

Buy a cheap 3 dollar set of metal tweezers. Flame sterilize. Rip open fruit pluck out a piece. Place on agar. Done


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OfflineQave
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Re: Tissue cloning sterile technique question [Re: metaphoric]
    #26567013 - 03/30/20 11:07 AM (3 years, 9 months ago)

Quote:

metaphoric said:
How many times have you used this tek and have you ever had it contaminate? I've stayed away from LCs so far because I hate how you don't know if you're contaminated until months later when you try to fruit.




I only use the needle biopsy method from that tek but he's using the little 1/2 pint mason jars specifically to watch for contams. I prefer to use the needle biopsies in two ways.

1. Fill the syringe with distilled water, squirt each "plug" onto an agar plate. (sterilize everything before starting, and flame sterilize the needle between biopsies)

2. Start with a half-full syringe of LC medium, pull the plug all the way into the syringe to make an LC syringe. Sometimes it'll just shoop in on it's own, sometimes you gotta help it by sucking up a little more LC medium. I like to use a 1% LME solution to try and starve the bugs. After a week take whichever syringe looks best and squirt a mL into a real LC jar.


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Offlinemetaphoric
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Re: Tissue cloning sterile technique question [Re: Qave]
    #26567034 - 03/30/20 11:17 AM (3 years, 9 months ago)

Quote:

Qave said:
2. Start with a half-full syringe of LC medium, pull the plug all the way into the syringe to make an LC syringe. Sometimes it'll just shoop in on it's own, sometimes you gotta help it by sucking up a little more LC medium. I like to use a 1% LME solution to try and starve the bugs. After a week take whichever syringe looks best and squirt a mL into a real LC jar.




Have you ever considered shooting the biopsy directly onto some grains?


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:mushroom::mushroom::mushroom::mushroom::mushroom::mushroom::mushroom::mushroom::mushroom::mushroom:
:mushroom:                  :mushroom2:                :mushroom:
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:mushroom:      :mushroom2::mushroom2::mushroom2::mushroom2::mushroom2:      :mushroom:
:mushroom::cookiemonster: BEHOLD :elmo::mushroom:
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OfflineQave
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Re: Tissue cloning sterile technique question [Re: metaphoric]
    #26567063 - 03/30/20 11:35 AM (3 years, 9 months ago)

Nah, agar and 2 oz souffle cups are super cheap so there's no reason not to clone to agar.


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Offlinemetaphoric
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Re: Tissue cloning sterile technique question [Re: Qave]
    #26567094 - 03/30/20 11:48 AM (3 years, 9 months ago)

Quote:

Qave said:
Nah, agar and 2 oz souffle cups are super cheap so there's no reason not to clone to agar.




What's your initial contam rate? Do your biopsies generally start off clean?


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OfflineWolfenstein
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Re: Tissue cloning sterile technique question [Re: metaphoric]
    #26567363 - 03/30/20 01:43 PM (3 years, 9 months ago)

I've had success from this. Used nicest fruit from first flush of first tub to make a quart which I then g2g'd to 14 quarts. The resulting two tubs were markedly more productive.

Mighta been total chance though, the first tub was MS.


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Edited by Wolfenstein (03/30/20 01:44 PM)


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Invisibleeatyualive
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Re: Tissue cloning sterile technique question [Re: Wolfenstein]
    #26567383 - 03/30/20 01:53 PM (3 years, 9 months ago)

I use an exacto. I would not leave a cup of alcohol sitting in the sab. You want to flame outside the sab. Also a bernzomatic torch makes things nice and easy. I never leave anything sitting in alcohol. It’s not necessary. The bernzo torch will burn everything off the blade. And you don’t have the chance of accidentally knocking over a cup of alcohol while your flaming your tools.

I use the mini scissors like you are talking about for cutting fruits for prints. The thickness of the metal takes time to sterilize. Sometimes 10-20 seconds. A thin scalpel or exacto blade is about 1-2 seconds. This may not mean much now, but when you start doing more clones and culturing it will make a hell of a lot of difference.

Bernzo and exacto


You can also simply sterilize your utensil wrapped in foil. Unwrap right before you take the cut. But if you flaw you’re technique now you have to resterilize in the pc. The alcohol will only sanitize.


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Edited by eatyualive (03/30/20 02:02 PM)


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OfflineA.k.aM
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Re: Tissue cloning sterile technique question [Re: eatyualive] * 1
    #26567626 - 03/30/20 03:46 PM (3 years, 9 months ago)

I use the same thing, scalpel and culinary torch.

I do have tweezers and forceps but they take soooo long to heat and cool, and get hot as fuck.


There’s a million good ways to do it, I’m just a fan of something I can do on a whim in 30 seconds.


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